I. Field of the Invention
This invention is directed to a method of enhancing the biological activity of vascular endothelial growth factors (VEGF). The invention further concerns certain VEGF variants having enhanced biological activity. The invention also concerns methods and means for preparing these variants, and pharmaceutical compositions comprising them. The invention further concerns methods of treatment using, and articles of manufacture containing such VEGF variants.
II. Description of Background and Related Art
Vascular endothelial growth factor (VEGF), also referred to as vascular permeability factor (VPF), is a secreted protein generally occurring as a homodimer and having multiple-biological functions. The native human VEGF monomer occurs as one of five known isoforms, consisting of 121, 145, 165, 189, and 206 amino acid residues in length after removal of the signal peptide. The corresponding homodimer isoforms are generally referred to as hVEGF121, hVEGF145, hVEGF165, hVEGF189, and hVEGF206, respectively. The known isoforms are generated by alternative splicing of the RNA encoded by a single human VEGF gene that is organized in eight exons, separated by seven introns, and has been assigned to chromosome 6p21:3 (Vincenti et al., Circulation 93:1493-1495 [1996]). A schematic representation of the various forms of VEGF generated by alternative splicing of VEGF mRNA is shown in FIG. 1, where the protein sequences encoded by each of the eight exons of the VEGF gene are represented by numbered boxes. VEGF165 lacks the residues encoded by exon 6, while VEGF121 lacks the residues encoded by exons 6 and 7. With the exception of hVEGF121, all VEGF isoforms bind heparin. The lack of a heparin-binding region in hVEGF121 is believed to have a profound effect on its biochemical properties. In addition, proteolytic cleavage of hVEGF produces a 110-amino acid species (hVEGF110).
hVEGF121 and hVEGF165 are the most abundant of the five known isoforms. They both bind to the receptors KDR/Flk-1 and Flt-1 but hVEGF165-additionally binds to a more recently discovered receptor (VEGF165R) (Soker et al., J. Biol. Chem. 271:5761-5767 [1996]). VEGF165R has been recently cloned by Soker et al., and shown to be equivalent to a previously-defined protein known as neuropilin-1 (Cell 92:735-745 [1998]). The binding of hVEGF165 to the latter receptor is mediated by the exon-7encoded domain, which is not present in hVEGF121.
VEGF is a potent mitogen for micro- and macrovascular endothelial cells derived from arteries, veins, and lymphatics, but shows significant mitogenic activity for virtually no other normal cell types. The denomination of VEGF reflects this narrow target cell specificity. VEGF has been shown to promote angiogenesis in various in vivo models, including, for example, the chick chorioallantoic membrane (Leung et al., Science 246:1306-1309 [1989]; Plouet et al., EMBO J 8:3801-3806 [1989]); the rabbit cornea (Phillips et al., In Vivo 8:961-965 [1995]); the primate iris (Tolentino et al., Arch Opthalmol 114:964-970 [1996]); and the rabbit bone (Connolly et al., J. Clin. Invest. 84:1470-1478 [1989]). As a result of its pivotal role in angiogenesis (spouting of new blood vessels) and vascular remodeling (enlargement of preexisting vessels), VEGF is a promising candidate for the treatment of coronary artery disease and peripheral vascular disease. High levels of VEGF are expressed in various types of tumors in response to tumor-induced hypoxia (Dvorak et al., J. Exp. Med. 174:1275-1278 [1991]; Plate et al., Nature 359:845-848 [1992]), and tumor growth has been inhibited by anti-VEGF antibodies and soluble VEGF receptors (Kim et al., Nature 362:841-844 [1993]; Kendall and Thomas, PNAS USA 90:10705-10709 [1993]).
The biologically active form of hVEGF121 is a homodimer (in which the two chains are oriented anti-parallel) containing one N-linked glycosylation site per monomer chain at amino acid position 75 (Asn-75), which corresponds to a similar glycosylation site at position 75 of hVEGF165. If the N-linked glycosylation structures are removed, the biologically active molecule has a molecular weight of about 28 kDa with a calculated pI of 6.1. Each monomer chain in the hVEGF121, homodimer has a total of nine cysteines, of which six are involved in the formation of three intra-chain disulfides stabilizing the monomeric structure, two are involved in two inter-chain disulfide bonds stabilizing the dimeric structure, while until recently one cysteine (Cys-116) has been believed to remain unpaired. Recently, a Cys(116)xe2x80x94Cys(l 16) inter-chain disulfide bond has been reported in E. coli derived recombinant hVEGF121 (Keck et al., Arch. Biochem. Biophys. 344:103-113 [1997]), and there are data indicating that VEGF121, as produced in nature, also occurs in the form of homodimers that have the cysteines at positions 116 disulfide-bonded with each other. EP 0 484 401 describes the substitution of one or more cysteine residues, including Cys-116, within the native VEGF molecule by another amino acid, to render the molecule more stable.
The present invention concerns methods and means for enhancing the biological activity of vascular endothelial growth factor (VEGF), new VEGF variants with enhanced biological activity, and various uses of such new variants.
In a specific aspect, the invention concerns a method of enhancing the biological activity of a VEGF originally having a cysteine (C) residue at a position corresponding to amino acid position 116 of the 121 amino acids long native mature human VEGF (hVEGF)21) by removing such cysteine (C) residue to produce a VEGF variant. The variant preferably comprises a glycosylation site at a position corresponding to amino acid positions 75-77 of hVEGF121, which is altered or removed, preferably by amino acid substitution within the glycosylation site to which the glycosylation would normally attach, so that glycosylation can no longer occur.
In another aspect, the invention concerns a variant of a native VEGF that originally has a cysteine (C) residue at amino acid position 116 and a glycosylation site at amino acid positions 75-77, comprising the substitution of said cysteine (C) by another amino acid and having the glycosylation site altered or removed, wherein the amino acid numbering follows the numbering of the 121 amino acids long native human VEGF (hVEGF121), and wherein the variant has enhanced biological activity compared to hVEGF121. The invention also concerns nucleic acid encoding such VEGF variants, a vector comprising the nucleic acid, cells transformed with such vector, and method for making the novel VEGF variants.
In yet another aspect, the invention concerns a composition comprising a VEGF variant having a cysteine (C) residue at amino acid position 116 substituted by another amino acid, and a glycosylation site at amino acid positions 75-77 altered or removed, wherein the amino acid numbering follows the numbering of the 121 amino acids long native human VEGF (hVEGF121).
In a further aspect, the invention concerns a method of inducing angiogenesis and/or vascular remodeling by administering to a patient in need a VEGF variant having a cysteine (C) residue at amino acid position 116 substituted by another amino acid, and a glycosylation site at amino acid positions 75-77 altered or removed, wherein the amino acid numbering follows the numbering of the 121 amino acids long native human VEGF (hVEGF121). In a particular embodiment, this method concerns the treatment of coronary artery disease or peripheral vascular disease.
In a still further aspect, the invention concerns a method for the prevention or repair of injury to blood vessels by administering an effective amount of a VEGF variant having a cysteine (C) residue at amino acid position 116 substituted by another amino acid, and a glycosylation site at amino acid positions 75-77 altered or removed, wherein the amino acid numbering follows the numbering of the 121 amino acids long native human VEGF (hVEGF121). In a particular embodiment, the injury is associated with microvascular angiopathy, such as thrombotic microangiopathy (TMA). In a further embodiment, the invention concerns the treatment of microvascular angiopathy, e.g. TMA of the kidney, heart, or lungs. In a particularly preferred embodiment, the invention concerns the prevention or repair of injury to blood vessels in association with hemolytic uremic syndrome (HUS), including thrombotic thrombocytopenic purpura (TTP).
In another aspect, the invention concerns a method for the treatment of essential hypertension by administering an effective amount of a VEGF variant having a cysteine (C) residue at amino acid position 116 substituted by another amino acid, and a glycosylation site at amino acid positions 75-77 removed, wherein the amino acid numbering follows the numbering of the 121 amino acids long native human VEGF (hVEGF121).
In a different aspect, the invention concerns an article of manufacture comprising a VEGF variant as hereinbefore defined, a container, and a label or package insert with instructions for administration.
In all embodiments, the VEGF variant preferably is N75Q,C116S hVEGF121.